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Validation of affinity reagents using antigen microarrays
KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).ORCID iD: 0000-0003-1363-5796
KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO).
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2011 (English)In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 29, no 5, 555-563 p.Article in journal (Refereed) Published
Abstract [en]

There is a need for standardised validation of affinity reagents to determine their binding selectivity and specificity. This is of particular importance for systematic efforts that aim to cover the human proteome with different types of binding reagents. One such international program is the SH2-consortium, which was formed to generate a complete set of renewable affinity reagents to the SH2-domain containing human proteins. Here, we describe a microarray strategy to validate various affinity reagents, such as recombinant single-chain antibodies, mouse monoclonal antibodies and antigen-purified polyclonal antibodies using a highly multiplexed approach. An SH2-specific antigen microarray was designed and generated, containing more than 6000 spots displayed by 14 identical subarrays each with 406 antigens, where 105 of them represented SH2-domain containing proteins. Approximately 400 different affinity reagents of various types were analysed on these antigen microarrays carrying antigens of different types. The microarrays revealed not only very detailed specificity profiles for all the binders, but also showed that overlapping target sequences of spotted antigens were detected by off-target interactions. The presented study illustrates the feasibility of using antigen microarrays for integrative, high-throughput validation of various types of binders and antigens.

Place, publisher, year, edition, pages
2011. Vol. 29, no 5, 555-563 p.
Keyword [en]
protein microarray, antibody validation, affinity reagent, antigen, specificity, SH2
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-48322DOI: 10.1016/j.nbt.2011.11.009ISI: 000305606500007Scopus ID: 2-s2.0-84862014285OAI: oai:DiVA.org:kth-48322DiVA: diva2:457333
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceKnut and Alice Wallenberg Foundation
Available from: 2011-11-17 Created: 2011-11-17 Last updated: 2017-12-08Bibliographically approved
In thesis
1. Protein microarrays for validation of affinity binders
Open this publication in new window or tab >>Protein microarrays for validation of affinity binders
2011 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Is specificity an important issue regarding affinity reagents? What about the validation of affinity reagents today, is it good enough? This depends on the application and the producer of the reagent. Validation should be the most important marketing argument that can be found.Today there is a continuous growth of both the number of affinity reagents that are produced and the different types of affinity reagents that are developed. In proteomics they become more and more important in exploring the human proteome. Therefore, validated affinity reagents should be on top of every proteomic researcher’s list. How should this be accomplished?Better international agreements on how affinity reagents should be tested to be regarded as functional reagents are needed. One of the most important issues is the specificity of the affinity reagent. An international standard for which specific validation that is needed for different kinds of applications would be very useful.In this thesis, it is shown that the protein microarray platform that was established within the HPA project at KTH is a very good tool to determine the specificity of different affinity binders.In the first study, the production of mono-specific antibodies for tissue profiling in the Human Protein Atlas (HPA) project is presented. The section describing the use of protein microarrays for validation of the antibodies is relevant for this thesis. The implementation of protein microarrays in the HPA workflow was an important addition, because a deeper insight of the specificity of all the antibodies produced were now available.In a second study, bead based arrays were compared to planar protein microarrays used in the HPA project. In this study, 100 different bead identities were coupled with 100 different antigens and mixed together to generate an array. The correlation between the two types of assays was very high and the conclusion was that the methods can be used as backup to each other.A third study was a part of an international initiative to produce renewable affinity binders against proteins containing SH2 domain. Here, the HPA protein microarrays were modified to analyze different types of reagents produced at six laboratories around the world. Monoclonal antibodies, single chain fragment and fibronectin scaffolds were tested as well as mono-specific antibodies. It was shown to be possible to adapt protein microarrays used in the HPA project to validate other kinds of affinity reagents.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2011. 31 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2011:23
Keyword
Microarray, protein, antibody, antigen, affinity, validation
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-48256 (URN)978-91-7501-149-3 (ISBN)
Presentation
2011-12-09, FB42, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 10:00 (Swedish)
Opponent
Supervisors
Projects
Development and applications of protein microarraysThe Swedish Human Proteome Resource (HPR) program
Note
QC 20111117Available from: 2011-11-17 Created: 2011-11-16 Last updated: 2011-11-17Bibliographically approved
2. On Generation and Applications of High-Density Protein Microarrays
Open this publication in new window or tab >>On Generation and Applications of High-Density Protein Microarrays
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Affinity proteomics has experienced rapid development over the last two decades and one of the most promising platforms to emerge are the protein microarrays. The combination of affinity reagents and miniaturisation enables assays for simultaneous high throughput and sensitive protein analysis. Due to the combination of these desrable properties, a multitude of protein array platforms for rapid and efficient study of proteomes and protein interactions are in use today. Although the protein microarray field has more than two decades of history to look back on the development of new protein microarray platforms continues to this day and beyond.

In the paper I in this thesis, a microarray of eluates from dried blood spot samples collected from neonates were designed and utilised for detection of complement factor 3 (C3) deficiency. The data acquired from the microarrays platform were compared to C3 levels obtained through enzyme-linked immunosorbant assay (ELISA), and the microarray assay were found to separate the C3 deficient samples from the controls. The conclusion of this investigation was that the microarray platform would be suitable for high-throughput screening of C3 deficiency in neonates. Paper II outlines the work in developing a multiplex platform for validation of affinity reagents. A set of 398 affinity binders, originating from five research groups, were profiled against 432 antigens and representing both polyclonal rabbit antibodies, monoclonal mouse antibodies, and recombinant single-chain variable fragments. Approximately 50% of the binders were found to preferably recognise their intended target while 10% of the binders did not generate any, or low, signals with their respective targets. For paper III, a reverse phase array (RPPA) platform using fluorescence-based detection of IgA deficiency in over 2.000 samples where validated on a label-free detection system and ELISA. The data from the label-free platform and the RPPA were found agree well with each other while data from ELISA did with neither of them. It was found that the label-free platform proved to be well-suited for detection of IgA in serum. Paper IV describes one of the world’s largest protein microarrays containing 21.120 recombinant protein fragments. We describe some of the possible applications of these large-scale arrays, such as binding profiles for the validation of antibodies with 11.520 and 21.120 recombinant proteins, as well as screening for autoimmunity in human serum samples.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2015. ix, 63 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2015:12
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-168165 (URN)978-91-7595-579-7 (ISBN)
Public defence
2015-06-12, Inghe-salen, Tomtebodavägen 18a, Solna, 10:00 (English)
Opponent
Supervisors
Note

QC 20150528

Available from: 2015-05-28 Created: 2015-05-27 Last updated: 2016-01-28Bibliographically approved

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Schwenk, Jochen M.Uhlén, Mathias

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